The Role Of MAP2K3 In Inflammatory Injury Induced By DON And Screening And Validation Of Its Inhibitors

Deoxynivalenol(DON)is a class of monotrichosporene toxins mainly produced by Fusarium graminae and Fusarium flavus.DON is the most common trichosporene toxin in cereals and there is serious pollution in our country and even in the world.Once people and animals eat DON contaminated grain and food,it will not only cause acute toxicity such as nausea,vomiting and diarrhea,but also lead to decreased appetite,slower growth rate,reduced utilization rate of animal feed,gastrointestinal mucosa damage and other toxicity,which brings great challenges to the development of animal husbandry.The cytotoxicity,growth inhibition and immunotoxicity caused by DON have been thoroughly revealed,and the important role of signaling pathways in the toxic damage caused by DON has been revealed.However,the key toxic genes or targets of DON induced inflammatory injury remain to be further elucidated.Although oxidative stress antagonists can effectively reduce part of the toxic effects of DON on cells and animals,they have disadvantages of poor specificity and low antagonistic ability due to their non-specific antagonists.Therefore,it is still important to find specific antagonists targeting inflammatory damage caused by DON.With the continuous development of molecular biology technology,target-based screening of drugs is the main method for the pharmaceutical industry to find new drugs.Therefore,in our previous study,J774 A.1 cells were used as the model,and CRISPR-Cas9 was used to screen the whole genome to find the key target of DON.It was found that the cell line with MAP2K3 gene deletion could effectively resist the toxic effect caused by DON,and MAP2K3 was speculated to be the key target of DON inflammatory injury.In this study,the mechanism of MAP2K3 in inflammatory injury induced by DON was studied,and the key role of MAP2K3 targets was positively and negatively verified at the cellular level from three aspects of inflammation,oxidative stress and apoptosis,and the specific inhibitors of MAP2K3 protein were virtually screened.Then,the toxicity of small molecule inhibitors against DON was verified from three aspects of inflammation,oxidative stress and apoptosis,and the key amino acid residues at the binding site were further verified,and the toxicity of drugs against DON was further verified at the animal level.The following results were obtained:1.MAP2K3 plays a key role in inflammatory response induced by DON.Western blotting(Western blot)showed that 1 μ M,2 μ M and 4 μ M DON significantly promoted the expression of IL-6,IL-1 β,TNF-α inflammatory factors within1 hour,and promoted the phosphorylation of p38,JNK,ERK1/2,p65,resulting in cellular inflammatory response.After interfering with MAP2K3 by siRNA,the expression of IL-6,IL-1 β and TNF-α was down-regulated,and the phosphorylation degree of p38,JNK,ERK1/2 and p65 was decreased.After overexpression of MAP2K3,the expression of IL-6,IL-1 β and TNF-α was up-regulated,and the phosphorylation of p38,JNK,ERK1/2 and p65 was increased.It is suggested that DON mainly activates the downstream p38 pathway through MAP2K3 to lead to inflammatory response.When interfering with MAP2K3,the degree of phosphorylation of p38 decreases and the inflammatory response of cells also decreases.After overexpression of MAP2K3,the degree of phosphorylation of p38 is enhanced,and the inflammatory response of cells is also aggravated.This suggests that MAP2K3 plays a key role in DON-induced inflammation.2.MAP2K3 plays a key role in oxidative stress induced by DONIn order to study the effects of different concentrations of DON on oxidative stress,the contents of ROS,MDA,SOD,GSH,NRF-2,NRF-1,p62 and HO-1 were detected by flow cytometry,enzyme labeling and Westernblot,and the morphology of mitochondria was observed by transmission electron microscope.The results showed that different concentrations of DON significantly increased the contents of ROS and MDA,and downregulated the contents of SOD and GSH.DON of 2 μ M and 4 μ M significantly downregulated the expression of NRF-2,NRF-1,p62 and HO-1,and DON of 1 μ M and 2 μ M could lead to mitochondrial swelling,crest disappearance and Golgi vacuolization,indicating that different concentrations of DON caused oxidative damage.After siRNA interference with MAP2K3,the content of ROS and MDA decreased,the content of SOD and GSH increased,the expression of NRF-2,NRF-1,p62 and HO-1 increased,and the mitochondrial swelling,crest disappearance and Golgi vacuolization induced by DON were reversed,indicating that interference with MAP2K3 can alleviate the oxidative damage induced by DON.After overexpression of MAP2K3,the content of ROS and MDA increased,the content of SOD and GSH decreased,and the expression of NRF-2,NRF-1,p62 and HO-1 decreased,indicating that overexpression of MAP2K3 can further induce oxidative damage induced by DON.This shows that MAP2K3 plays a key role in the oxidative damage caused by DON.3.MAP2K3 plays a key role in apoptosis induced by DON.In order to study the effect of different concentrations of DON on apoptosis,the expression and activation of apoptosis-related proteins in Bax,Bim,Bcl-2,p53,Caspase 3,Caspase 8 and Caspase 9 were detected by flow cytometry,fluorescence microscope and Westernblot.The results showed that different concentrations of DON significantly induced apoptosis,up-regulated the expression of Bim and down-regulated the ratio of Bcl-2/Bax,promoted the expression of p53 protein,and promoted the activation of Caspase 3,Caspase8 and Caspase 9 to play the role of apoptosis.After siRNA interference with MAP2K3,WB results showed that the expression of Bim decreased,the ratio of Bcl-2/Bax increased,the expression of p53 decreased,the activation of Caspase 3 and Caspase 9 decreased,and the proportion of apoptosis decreased significantly,indicating that interfering with MAP2K3 could significantly alleviate the apoptosis induced by DON.After overexpression of MAP2K3,the expression of Bim increased,the ratio of Bcl-2/Bax decreased,the expression of p53 increased,the activation of Caspase 3,Caspase 8 and Caspase 9 increased,and the proportion of apoptosis increased significantly,indicating that overexpression of MAP2K3 can significantly induce apoptosis induced by DON.This suggests that MAP2K3 plays a key role in DON-induced apoptosis.4.Ole can inhibit the expression of MAP2K3,thus antagonizing the injury of inflammation,oxidative stress and apoptosis induced by DON.In order to screen inhibitors of MAP2K3 protein to antagonize the inflammatory injury induced by DON,MAP2K3 protein was modeled by SWISSMODEL.The crystal structure of MAP2K3 was docked with prepared small molecules by SYBYL2.0 software.The results showed that the top four were methyl hesperidin,Ole,chebulic acid and quercetin.Westernblot test showed that methyl hesperidin and Ole could inhibit the expression of MAP2K3 protein.And CCK-8 detection of methyl hesperidin and Ole had very low cytotoxicity,but only Ole could alleviate the cytotoxicity induced by DON.The effect of Ole was verified at the cellular level,and the results showed that 20 μM,40 μM and 60 μM Ole could reverse the cell inflammation,oxidative stress and apoptosis induced by DON.Furthermore,through CETSA and DARST tests,it was found that Ole exerted its inhibitory effect by directly combining with LYS149,SER194 and TYR230 amino acids.At the animal level,Ole can alleviate the loss of appetite and body weight loss caused by DON,reduce MDA,increase the contents of SOD,GSH,NRF-2,NRF-1 and HO-1,and alleviate liver oxidative damage.In short,Ole inhibits its expression by binding to MAP2K3 protein,thus reversing DON-induced cell inflammatory injury and animal growth toxicity and hepatotoxicity.